Arenaviruses are responsible for severe hemorrhagic fevers with high morbidity and mortality worldwide. In the absence of vaccines or specific therapies, these viruses are recognized as Category A priority pathogens that pose significant threats to public health and biodefense. Intervention strategies that target the arenavirus envelope glycoprotein complex (GPC) and virus entry into the host cell hold promise for combating these lethal infections. Unlike other Class I viral fusion proteins, GPC contains three subunits: GP1, GP2 and a unique stable signal peptide (SSP). We have demonstrated that SSP acts in conjunction with the GP2 fusion subunit to sense acidic pH in the maturing endosome and thereby trigger the structural transitions leading to virus-cell membrane fusion. We have characterized six chemically distinct classes of small-molecule arenavirus fusion inhibitors that differ in their selectivities against New World (NW) and Old World (OW) arenaviruses, but share a common binding site on GPC. Our genetic studies suggest that these compounds bind at the pH-sensing interface of SSP and GP2. In preliminary studies, we show that photoaffinity derivatives of one such inhibitor, lassamycin-1, can specifically label SSP and GP2 subunits in recombinant Lassa virus (LASV) GPC purified from insect-cell membranes, consistent with inhibitor binding at the SSP-GP2 interface. Thus, we are uniquely positioned to dissect the molecular basis of arenavirus fusion activation and its inhibition. We will accomplish these objectives by using biochemical, pharmacological, genetic and state-of-the-art mass spectrometry approaches to pursue the following aims: (1) Identify photolabeled amino-acid residues in LASV GPC to elucidate the inhibitor-binding site. We will utilize combined chemical biology and high-resolution tandem mass spectrometric sequencing to identify amino-acid residues in GPC modified by lassamycin derivatives. Labeled SSP and GP2 subunits and peptides will be enriched for mass spectrometry using a stable-isotope- tagged, cleavable biotin linker and click chemistry to functionalize the covalently bound inhibitor. New inhibitors with photolabile groups at other sites will be used to identify residues elsewhere in the binding pocket. (2) Identify and characterize the homologous inhibitor-binding site in JUNV GPC. Lassamycin derivatives also show inhibitory activity against the NW Junn (JUNV) virus, and we will use mass spectrometry to identify the homologous inhibitor-binding site in JUNV GPC. (3) Develop a structural model of the interfacial surfaces of SSP and GP2 to identify common and species-specific determinants of fusion activation and its inhibition. As structural information on intact GPC is not available, we will utilize our results to construct a spatial model of the SSP-GP2 interface. Scanning mutagenesis and functional assays will be employed to investigate the role of conserved and divergent amino-acid sidechains in controlling GPC fusion activity and inhibitor selectivity. These studies will enhance our understanding of the molecular basis for pH sensing and fusion activation in GPC, and how drug-like molecules can interfere in this process.